The invention relates in general to a separation column, and, in particular, to a bonded phase photopolymerized sol-gel column.
Capillary electrochromatography (CEC) has been regarded as a very promising analytical separation technique that combines the efficiency of capillary zone electrophoresis (CZE) with the selectivity of liquid chromatography. Although CEC has been applied in many different areas, packed-column preparation and low-detection sensitivity remain challenges of this technique. Capillary columns containing small silica packings have been the mainstay of CEC. One disadvantage of packed columns is the fabrication of porous frits of controlled pore sizes, lengths, and high mechanical stabilities. In response to frit fabrication problems, surface-functionalized open-tubular capillary columns and monolithic capillaries are being developed as variants of packed capillary columns. Monolithic capillary columns have received much attention because of the advantages offered in the control of permeability and surface charge.
A major challenge in CEC techniques is the detection of samples containing analytes at low concentration. The lack of sensitivity at low concentration stems from the small sample volume and the short optical path length for on-line detection. Dedicated sample preparation schemes that enrich the target analytes before sample injection are often necessary in order to obtain the necessary sensitivity for many real-world analyses. Schemes such as solvent-solvent extraction and solid-phase extraction are often very tedious and time-consuming.
An alternative to these schemes is on-line preconcentration. In gas chromatography, this goal is met by passing a gas stream through a cold column that is subsequently heated. In high-performance liquid chromatography (HPLC), this process is usually done by gradient HPLC in which the analytes are retained on the column much more strongly for the first solvent than for succeeding ones. On-line preconcentration has also enjoyed some success in electrophoretic separations. For example, in capillary electrophoresis (CE), these include isotachophoresis, sample stacking, sweeping, and the use of a dynamic pH junction. In CZE, F. E. P. Mikkers, F. M. Everaerts, P. E. M. Verheggen, J. Chromatogr. 169 (1979), pp. 1-10 and R. L. Chien, D. S. Burgi, Anal. Chem. 64 (1992) pp. 489A-496A, demonstrated that changes in electric field strength between sample and background solution zones can focus (i.e., stack) charged species. In electrokinetic chromatography, J. P. Quirino, S. Terabe, Science, 282 (1998) pp. 465-68 and J. P. Quirino, S. Terabe, Anal. Chem. 71(8) (1999) pp. 1638-44, have shown that micelles can act to concentrate (i.e., sweep) neutral and charged species.
In CEC using particle (e.g., octadecyl silica) packed columns, focusing effects similar to that in gradient high performance liquid chromatography have been reported. These focusing effects were achieved using (1) step-gradient elution, (2) preparation of the sample in a noneluting solvent, or (3) injection of a water plug after sample injection. In M. R. Taylor, P. Teale, D. Westwood, D. Perrett, Anal. Chem. 69 (1997) pp. 2554-58, the authors were the first to report the use of a step-gradient for the preconcentration of steroidal samples in 1997. In D. A. Stead, R. G. Reid, R. B. Taylor, J. Chromatogr. A 798 (1998) pp. 259-67, the authors achieved a 17-fold increase in the detection sensitivity of a mixture of steroids by preconcentration using a noneluting sample matrix. In Y. Zhang, J. Zhu, L. Zhang, W. Zhang, Anal. Chem. 72 (2000) pp. 5744-47, the authors also used a noneluting solvent for the preconcentration of benzoin and mephenytoin by a factor of 134 and 219, respectively. In C. M. Yang, Z. El Rassi, Electrophoresis 20 (1999) pp. 2337-42, the authors reported on the preconcentration of a dilute sample of pesticides using a short plug of water injected after a long plug of sample. In M. J. Hilhorst, G. W. Somsen, G. J. de Jong, Chromatographia 53 (2001) pp. 190-96, the authors demonstrated preconcentration of structurally related steroids using a noneluting matrix and step-gradient elution. A gain in sensitivity of 7 to 9 times was reported. Similarly, in T. Tegeler, Z. El Rassi, Anal. Chem. 73 (14) (2001) pp. 3365-72, the authors just recently reported preconcentration of analytes in a mixture of carbamate insecticides using a combination of a noneluting matrix and step-gradient elution. The maximum allowable sample plug length was ˜20 cm and a 500-fold sensitivity increase is achieved for carbofuran. Zhang and co-workers achieved a further increase in detection sensitivity by combining field-enhanced sample injection with solvent gradient elution. They demonstrated a 17,000-fold increase in peak height for a positively charged analyte, propatenene.
It is desirable to provide a separation column with improved characteristics and that is easy to make.